ASSEMBLY OF THE DIVISION SEPTUM IN ESCHERICHIA COLI

我们工作的长期重点是细菌的细胞分裂。特别是，我们感兴趣的是分裂隔膜是如何形成的，以及它的形成是如何与细胞周期中的其他事件如染色体分离相协调的。一个辅助目标是了解蛋白质是如何定位到特定的亚细胞位点。这种兴趣源于观察到，许多蛋白质参与隔膜组装定位于分裂网站。细胞分裂和蛋白质定位是对包括人类在内的所有生物体都很重要的基本细胞过程，并且是癌症等疾病的基础，但由于技术限制，通常难以研究。大肠杆菌中丰富的遗传工具使其成为研究基本细胞过程的理想模式生物。E.大肠杆菌需要至少九种必需蛋白质，所有这些蛋白质都定位于分裂位点。我们的假设是，这些蛋白质形成的复合物，包含所有的活动需要隔膜组装，这些蛋白质之间的相互作用是重要的，他们的招聘分裂网站和调节他们的活动。因此，我们想要更多地了解这些蛋白质的详细功能，以识别这些蛋白质之间的相互作用，并了解该集合是否完整。为了解决这些问题，我们将表征FtsI，一种具有与肽聚糖合成相关的酶活性（转肽酶）的膜蛋白。 具体来说，我们将（i）使用遗传学和荧光显微镜的组合来识别FtsI中将该蛋白质靶向分裂位点的序列;（ii）使用生物化学和遗传学方法来寻找与FtsI直接相互作用的蛋白质;和（iii）使用荧光显微镜确定假定参与隔肽聚糖合成并与FtsI相互作用的三种转糖基酶的亚细胞位置。更好地理解简单细菌系统中的细胞分裂可能会揭示其他生物体（包括人类）的这些过程。 此外，更好地了解这些过程可能会导致更多基于知识的方法来开发新的抗生素。在这方面，值得注意的是，这里要研究的四种蛋白质（FtsI和三种转糖基酶）是β-内酰胺抗生素的主要靶标。

The long-term focus of our work is cell division in bacteria. In particular, we are interested in how the division septum is formed, and how its formation is coordinated with other events in the cell- cycle such as chromosome segregation. An ancillary objective is to understand how proteins are localized to specific subcellular sites. This interest stems from the observation that many proteins involved in septum assembly are localized to the division site. Cell division and protein localization are fundamental cellular processes of importance to all organisms, including humans, and underlie diseases such as cancer, but are often difficult to study due to technical limitations. The wealth of genetic tools available in Escherichia coli makes this an ideal model organism for studies of basic cellular processes. Septum assembly in E. coli requires at least nine essential proteins, all of which localize to the division site. Our hypothesis is that these proteins form of complex that contains all of the activities need for septum assembly, and that interactions among these proteins are important for their recruitment to the division site and for regulating their activities. Thus, we want to know more about the detailed function of these proteins, to identify interactions among these proteins, and to know whether the set is complete. To approach these issues we will characterize FtsI, a membrane protein with an enzymatic activity (transpeptidase) related to peptidoglycan synthesis. Specifically, we will (i) use a combination of genetics and fluorescence microscopy to identify sequences in FtsI that target this protein to the division site; (ii) use biochemical and genetic approaches to look for proteins that interact directly with FtsI; and (iii) use fluorescence microscopy to determine the subcellular location of three transglycosylases postulated to be involved in synthesis of septal peptidoglycan and to interact with FtsI. A better understanding of cell division in a simple bacterial system might shed light on these processes in other organisms, including humans. In addition, a better understanding of these processes might lead to more knowledge-based approaches to developing new antibiotics. In this regard, it is worth noting that four of the proteins to be studied here (FtsI and the three transglycosylases) are primary targets of beta-lactam antibiotics.